The devR gene product is characteristic of receivers of two-component regulatory systems and is essential for heterocyst development in the filamentous cyanobacterium Nostoc sp. strain ATCC 29133.

Strain UCD 311 is a transposon-induced mutant of Nostoc sp. strain ATC C 29133 that is unable to fix nitrogen in air but does so under anoxic conditions and is able to establish a functional symbiotic association with the hornwort Anthoceros punctatus. These properties of strain UCD 311 are consistent with previous observations that protection against oxygen inactivation of nitrogenase is physiologically provided within A. punctatus tissue. Upon deprivation of combined nitrogen, strain UCD 311 clearly differentiates heterocysts and contains typical heterocyst-specific glycolipids; it also makes apparently normal akinetes upon phosphate starvation. Sequence analysis adjacent to the point of the transposon insertion revealed an open reading frame designated devR. Southern analysis established that similar sequences are present in other heterocyst-forming cyanobacteria. devR putatively encodes a protein of 135 amino acids with high similarity to the receiver domains of response regulator proteins characteristics of two-component regulatory systems. On the basis of its size and the absence of other functional domains, DevR is most similar to CheY and Spo0F. Reconstruction of the mutation with an interposon vector confirmed that the transposition event was responsible for the mutant phenotype. The presence of wild-type devR on a plasmid in strain UCD 311 restored the ability to fix nitrogen in air. While devR was not essential for differentiation of akinetes, its presence in trans in Nostoc sp. strain ATCC 29133 stimulated their formation to above normal levels in aging medium. On the basis of RNA analysis, devR is constitutively expressed with respect to the nitrogen source for growth. The devR gene product is essential to the development of mature heterocysts and may be involved in a sensory pathway that is not directly responsive to cellular nitrogen status.     

Photosynthetic CO2 fixation and ribulose bisphosphate carboxylase/oxygenase activity of Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus.

The cyanobacterium Nostoc sp. strain UCD 7801, immediately after separation from pure cultures of a reconstituted symbiotic association with the bryophyte Anthoceros punctatus, exhibited a rate of light-dependent CO2 fixation that was eightfold lower than that measured in the free-living growth state. Ribulose bisphosphate carboxylase/oxygenase (RuBPC/O) specific activity was also eightfold lower in cell extracts of symbiotic strain 7801 relative to that in free-living cultures. The in vitro activity from symbiotic strain 7801 could not be increased by incubation under the standard RuBPC/O activation conditions. Polyclonal antibodies against the RuBPC/O large subunit were used in an enzyme-linked immunosorbent assay to determine that RuBPC/O accounted for 4.3 and 5.2% of the total protein in cell extracts of strain 7801 grown in symbiotic and free-living states, respectively. The results imply that the regulation of RuBPC/O activity in the symbiotic growth state is by a posttranslational mechanism rather than by an alteration in RuBPC/O protein synthesis. The amount of carboxyarabinitol bisphosphate required to irreversibly inhibit RuBPC/O activity of sybiotic cell extracts was 80% of that required for extracts of free-living cultures. This result indicates that any covalent modification of RuBPC/O in symbiotically associated Nostoc cells did not interfere with the ribulose bisphosphate binding site, since inactive enzyme also bound carboxyarabinitol bisphosphate.     

Nitrogen fixation by a marine non‐heterocystous cyanobacterium requires a heterotrophic bacterial consort

Cultures of the non‐heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro‐oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark‐mediated nitrogenase in the absence of DCMU nor light‐mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non‐axenic cultures of L. nodulosa. RT‐PCR demonstrated that this gene was expressed only in non‐axenic cultures. Western blotting showed that the Fe‐protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.     

Modes of reduction of nitrogen in heterocysts isolated from Anabaena species.

N2 fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, D-glucose 6-phosphate, 6-phosphogluconate and DL-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.     

The Relationships between Nitrogen Fixation and Several Kinds of Energy Metabolism by Gloeocapsa sp

Gloeocapsa sp., a species of anicellular blue-green alga, fixes dinitrogen mostly under light. The energy (ATP and reductant) needed for nitrogen fixation may be provided by photoreaction and aerobic catabolism. The nitrogenase activity (acetylene reduction) in vivo was decreased under the conditions of dark and inhibition of photo-phosphorylation or oxidative phosphorylation in the light. When photosystem Ⅱ was inhibited by the presence of DCMU, nitrogenase activities in both reactions of acetylene reduction and hydrogen evolution may be muchenhanced probably due to eliminating of the damage caused by the oxygen produced in the photolysis of water. The effects of the oxygen present in the atmosphere of the reaction systemand produced by the cells are different. It is shown that some trace oxygen seems to be required for nitrogen fixation by the energy supply of aerobic actabolism and oxidative phosphorylation. While the fixation of dinitrogen was inhibited by CO or no any reducible substrate was present, 70-100% of the energy accepted by nitrogenase was evolved as hydrogen. The algal cells also showed hydrogen uptake reaction, but no enhancement of nitrogen fixation by the hydrogen uptake was found.     

Regulation of expression of glutamine synthetase in a symbiotic Nostoc strain associated with Anthoceros punctatus.

A characteristic of N2-fixing cyanobacteria in symbiotic associations appears to be release of N2-derived NH4+. The specific activity of the primary ammonium-assimilating enzyme, glutamine synthetase (GS), was found to be three- to fourfold lower in Nostoc sp. strain 7801 grown in symbiotic association with the bryophyte Anthoceros punctatus than in free-living Nostoc sp. strain 7801. Quantitative immunological assays with antisera against GS purified from Nostoc sp. strain 7801 and from Escherichia coli indicated that similar amounts of the GS protein were present in symbiotic (50 micrograms mg-1) and free-living (68 micrograms mg-1) cultures. The conclusion from these experiments is that GS is regulated by a posttranslational mechanism in Anthoceros-associated Nostoc sp. strain 7801. However, the results of comparative catalytic and immunological experiments between N2- and NH4+-grown free-living Nostoc sp. strain 7801 implied control of GS synthesis. A correlation was not observed between the level of GS expression and the extent of symbiotic heterocyst differentiation in Nostoc sp. strain 7801 associated with A. punctatus.     

Liberation of ammonia during nitrogen fixation by a facultatively heterotrophic cyanobacterium

Nitrogen fixation (acetylene reduction) and ammonia liberation were studied in a facultatively heterotrophic cyanobacterium. Autotrophically grown cells lost acetylene reduction activity when incubated under anaerobic conditions; the activity was maintained in the presence of methionine sulfoximine; or by pretreatment of the cells with a carbon supply. Heterotrophically grown cells maintained acetylene reduction activity anaerobically in the absence of methionine sulfoximine. Both cell types required light for maintenance of activity. The data indicate that methionine sulfoximine preserves the intracellular pool of reductant needed for nitrogenase. Autotrophs and heterotrophs both liberated ammonia when treated with methionine sulfoximine under nitrogen-fixing conditions. However, on treatment with methionine sulfoximine under anaerobiosis, heterotrophs also accumulated large amounts of intracellular ammonia in a pool which was diminished by the Photosystem II inhibitor, 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). DCMU enhanced ammonia liberation without affecting acetylene reduction activity, and hence changed the ratio of acetylene reduced to ammonia formed by the heterotrophs. These data suggest a role for Photosystem II in ammonia liberation by the cyanobacteria.     

Regulatory effect of hydrogen on nitrogenase activity of the blue-green alga (cyanobacterium) Nostoc muscorum.

Preincubation of the blue-green alga (cyanobacterium) Nostoc muscorum under an atmosphere of argon plus acetylene in the light led to a greater than fourfold increase of light-induced hydrogen evolution and to a 50% increase of acetylene reduction, as compared to cells that had not been preconditioned. The basic and the increased hydrogen evolution were both due to nitrogenase activity. Furthermore, after preincubation the hydrogen uptake, usually observed with unconditional cells, was abolished. Nostoc preincubated under acetylene evolved hydrogen in the light even in the presence of nitrogen for at least 2 h, with a 15-fold increase as compared to the unconditioned cells. These acetylene effects could be completely abolished by the presence of hydrogen during acetylene preincubation. These findings indicate that the hydrogen concentration in N. muscorum cells plays a role in regulation of nitrogenase activity.     

Nitrogen fixation by Anabaena cylindrica

Hydrogen-supported nitrogenase activity was demonstrated in Anabaena cylindrica cultures limited for reductant. Nitrogen-fixing Anabaena cylindrica cultures sparged in the light with anaerobic gases in the presence of the photosynthesis inhibitor DCMU slowly lost their ability to reduce acetylene in the light under argon but exhibited near normal activities in the presence of 11% H₂ (balance argon). The hydrogen-supported nitrogenase activity was half-saturated between 2 and 3% H₂ and was strongly inhibited by oxygen (50% inhibition at about 5–6% O₂). Batch cultures of Anabaena cylindrica approaching stationary growth phase (“old” cultures) lost nitrogenase-dependent hydrogen evolution almost completely. In these old cultures hydrogen relieved the inhibitory effects of DCMU and O₂ on acetylene reduction. Our results suggest that heterocysts contain an uptake hydrogenase which supplies an electron transport chain to nitrogenase but which couples only poorly with the respiratory chain in heterocysts and does not function in CO₂ fixation by vegetative cells.     

PHYSIOLOGICAL ECOLOGY OF NITROGEN FIXING BLUE-GREEN ALGAL CRUSTS IN THE UPPER-SUBALPINE LIFE ZONE1

Blue-green algal (cyanobacterial) crusts composed of nitrogen fixing Nostoc commune Voucher ex Born. et Flah. and Tolypothrix conglutinata var. colorata Ghose were studied in the upper-subalpine life zone, Mission Mountain Wilderness, Montana. Rates of ethylene production were highest in the submerged shoreline crusts, lower for exposed crusts pioneering rocky shorelines and lowest in the Carex meadow. Nitrogenase activity (acetylene reduction technique) was constant between 200–285% crust moisture content (wet/dry weight) and then rapidly declined to 0 between 200–140%. Optimal temperatures for ethylene production by illuminated cells was between 20–30° C for T. conglutinata, 20° C for N. commune and about 25° C in darkness for both species. Nitrogenase activity by T. conglutinata in culture was unaffected by repeated freeze-thaw treatments whereas N. commune was severely inhibited. In contrast, N₂-ase activity of these two species in an intact crust was unaffected by repeated freeze-thaw treatments. Application of nitrogen-free growth medium to intact crusts increased nitrogenase activity by 3.7 times implying that these were mineral deficient under field conditions. Photosynthesis was light saturated at 125 μmol-m^?2.s^?1 whereas nitrogenase activity was light independent for cells with carbohydrate reserves. When carbohydrate reserves were reduced by 8 h incubation in darkness, between 1–3 h of illumination were required to restore nitrogenase activity to 80% of the maximum rate. Biochemical pathway inhibitor studies employing DCMU, MFA, and CCCP showed that oxidative metabolism was the source of reductant for acetylene reduction. Tetrazolium precipitation in heterocysts paralleled acetylene reduction activity in the inhibitor treated cells.     

Modes of reduction of nitrogenase in heterocysts isolated from Anabaena species

N₂ fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, d-glucose 6-phosphate, 6-phosphogluconate and dl-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.     

Response of nitrogenase to altered carbon supply in a Frankia-Alnus incana symbiosis

To study the effect of altered carbon supply on nitrogenase (EC 1.7.99.2), plants of Alnus incana (L.) Moench in symbiosis with the local source of Frankia were exposed to darkness for 2 days, and then returned to normal light/dark conditions. During the dark period nitrogenase activity in vivo (intact plants) and in vitro ( Frankia cells supplied with ATP and reductant), measured as acetylene reduction activity, was almost completely lost. Western blots for both the Fe-protein (dinitrogenase reductase) and the MoFe-protein (dinitrogenase) showed that, in particular, the amount of MoFe-protein was strongly reduced during darkness. Protein stained sodium dodecyl sulphate-polyacrylamide gels of Frankia protein showed that the nitrogenase proteins were the only abundant proteins that clearly decreased during darkness. During recovery, studied for 4 days, nitrogenase activity in vivo recovered to the level before dark treatment but was still only half of control activity, Nitrogenase activity in vitro and the amount of MoFe-protein, both expressed per Frankia protein, recovered and reached similar values in previously dark treated plants and in control plants. The rate of recovery was similar to the increase in activity of control plants, suggesting growth of Frankia in addition to synthesis of nitrogenase proteins during the recovery after carbon starvation.     

Nif- Hup- mutants of Rhizobium japonicum.

Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity.     

Induction of a nitrogenase activity rhythm inSynechococcus and the protection of its nitrogenase against photosynthetic oxygen

All oxygen levels are detrimental to the nitrogenase activity ofSynechococcus RF-1 cells. In continuous light, cultures maintain a high dissolved oxygen concentration and a continuous but usually low rate of nitrogenase activity.Cultures adapted to a light-dark regimen will reduce acetylene almost exclusively during the dark periods. When switched to continuous light, they continue to exhibit a diurnal rhythm in nitrogenase activity. While in continuous light, each upsurge of nitrogenase activity coincides with a marked drop in the net oxygen production rate; this drop is due largely to a concomitant increase in the dark respiration rate of the culture.The endogenous nitrogenase activity rhythm can be induced in continuous light by periodically lowering the oxygen concentration of the culture by either bubbling nitrogen through it or by treating the culture with 3(3,4-dichlorophenol)-1,1-dimethylurea (DCMU or diuron).     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.     

On the role of oxygen for nitrogen fixation in the marine cyanobacterium Trichodesmium sp

The marine, non-heterocystous, filamentous cyanobacterium Trichodesmium shows a distinct diurnal pattern of nitrogenase activity. In an attempt to reveal the factors that control this pattern, a series of measurements were carried out using online acetylene reduction assay. Light response curves of nitrogenase were recorded applying various concentrations of oxygen. The effect of oxygen depended on the irradiance applied. Above a photon irradiance of 16 mumol m(-2) s(-1) nitrogenase activity was highest under anoxic conditions. Below this irradiance the presence of oxygen was required to achieve highest nitrogenase activity and in the dark 5% oxygen was optimal. At any oxygen concentration a photon irradiance of 100 mumol m(-2) s(-1) was saturating. When Trichodesmium was incubated in the dark, nitrogenase activity gradually decreased and this decline was higher at higher levels of oxygen. The activity recovered when the cells were subsequently incubated in the light. This recovery depended on oxygenic photosynthesis because it did not occur in the presence of DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Recovery of nitrogenase activity in the light was faster at low oxygen concentrations. The results showed that under aerobic conditions nitrogenase activity was limited by the availability of reducing equivalents suggesting a competition for electrons between nitrogenase and respiration.     

Nitrogenase Activity and Photosynthesis in Plectonema boryanum

Nitrogen-starved Plectonema boryanum 594 cultures flushed with N(2)/CO(2) or A/CO(2) (99.7%/0.3%, vol/vol) exhibited nitrogenase activity when assayed either by acetylene reduction or hydrogen evolution. Oxygen evolution activities and phycocyanin pigments decreased sharply before and during the development of nitrogenase activity, but recovered in the N(2)/CO(2) cultures after a period of active nitrogen fixation. Under high illumination, the onset of nitrogenase activity was delayed; however, the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) eliminated this lag. Oxygen was a strong and irreversible inhibitor of nitrogenase activity at low (>0.5%) concentrations. In the dark, low oxygen tensions (0.5%) stimulated nitrogenase activity (up to 60% of that in the light), suggesting a limited but significant respiratory protection of nitrogenase at low oxygen tensions. DCMU was not a strong inhibitor of nitrogenase activity. A decrease in nitrogenase activity after a period of active nitrogen fixation was observed in the N(2)/CO(2-), but not in the A/CO(2-), flushed cultures. We suggest that this decrease in nitrogenase activity is due to exhaustion of stored substrate reserves as well as inhibition by the renewed oxygen evolution of the cultures. Repeated peaks of alternating nitrogenase activity and oxygen evolution were observed in some experiments. Our results indicate a temporal separation of these basically incompatible reactions in P. boryanum.     

Stimulation of nitrogenase activity and photosynthesis in some cyanobacteria by glyoxylate

Abstract The effect og glyoxylate on nitrogenase activity (C₂H₂ reduction) and photosynthesis (H¹⁴CO₃ fixation and O₂ evolution) was in vestigated in the three heterocystous cyanobacteria Anabaena cylindrica, A. variabiltis and N. muscorum. Glyoxylate had virtually no effect on the rate of dark respiration and was unable to sustain photoheterotrophic growth, though some slight stimulation (= 30%) of photorophic growth was noted. A considerable stimulation of both nitrogenase and photosynthetic activities was observed in presence of glyoxylate. In the light the stimulation increased with time up to about 15-25 h after adding optimal concentrations of 4–6 mM glyoxylate. Placing glyoxylate treated samples in the dark or adding DCMU (30 μM) in the light, showed that glyoxylate initially supported significantly higher nitrogenase activity than did samples in absence of glyoxylate. However, after a prolonged incubation in the dark or in presence of DCMU glyoxylate is unable to relieve the adverse effects of such conditions. The stimulation of the nitrogenase activity was even more pronounced when the glyoxylate was added to cells preincubated in the dark (“carbon starved”) than for cells kept constantly in light. The results suggest that glyoxylate, or a metabolite, may act as an inhibitor of cyanobacterial photorespiration and this hypothesis is discussed.     

Nitrogenase Activity in Cell-Free Extracts of the Blue-Green Alga, Anabaena cylindrica

Cell-free extracts with high nitrogenase activity were prepared by sonic oscillation and French press treatment from the blue-gree alga Anabaena cylindrica. Extracts were prepared from cells grown on a 95% N(2)-5% CO(2) gas mixture followed by a period of nitrogen starvation under an atmosphere of 95% argon-5% CO(2). No increase in the specific activity of extracts was achieved by breaking heterocysts. Activity (assayed by acetylene reduction) was found to be dependent on adenosine triphosphate (ATP), an ATP-generating system, and a low-potential reductant. Na(2)S(2)O(2) employed as reductant supports higher rates of nitrogenase activity than reduced ferredoxin. The activity is associated with a small-particle fraction that can be sedimented by ultracentrifugation. In contrast to the particulate nitrogenase of Azotobacter, which is stable in air, the A. cylindrica nitrogenase is an oxygen sensitive as nitrogenase prepared from anaerobic bacteria.     

Factors influencing dark nitrogen fixation in a blue-green alga.

Nitrogen-fixing activity declines first rapidly and then more gradually when Anabaenopsis circularis is transferred from light into dark conditions. The rate and duration of dark acetylene reduction (nitrogen fixation) depend upon conditions prevailing during the preceding light period. Factors (such as light intensity, CO2 concentration, and supply of glucose), which in the light affect photosynthesis and the accumulation of reserve carbon, have a profound effect on dark nitrogen fixation. Glucose greatly promotes nitrogen fixation in the light and supports prolonged nitrogenase activity in the dark. The results suggest that heterotrophic nitrogen fixation by blue-green algae in the field may be important both under light and dark conditions.